Anion exchange fractionation of serum proteins versus albumin elimination

Elimination of albumin, constituting more than 50% of total serum proteins, allows increased protein loads on immobilized pH gradient (IPG) gels and better visualization of low-abundance proteins; however, it may result in the loss of albumin-bound low-abundance proteins. In this study, we report the prefractionation of serum proteins by batch anion exchange chromatography into three fractions: one containing proteins with isoelectric points (pI values) higher than the pI of albumin, a second fraction containing proteins with pI values in the same range as the pI of albumin, and a third fraction containing proteins with pI values lower than the pI of albumin. This procedure uses common instrumentation, is carried out under denaturing conditions, and takes less than 30min. We also report the loss of a clinically established prostate cancer serum biomarker, prostate-specific antigen (PSA), after albumin is eliminated using two commercially available albumin elimination kits: one that uses Cibacron Blue F3GA, which achieves albumin depletion through dye-ligand binding, and one that uses specific albumin antibody. The loss of PSA secondary to albumin elimination exceeded that after batch anion exchange serum sample prefractionation.     

Strategies for the enrichment and identification of basic proteins in proteome projects

Two-dimensional gel electrophoresis (2-DE) is currently the method of choice for separating complex mixtures of proteins for visual comparison in proteome analysis. This technology, however, is biased against certain classes of proteins including low abundance and hydrophobic proteins. Proteins with extremely alkaline isoelectric points (pI) are often very poorly represented using 2-DE technology, even when complex mixtures are separated using commercially available pH 6-11 or pH 7-10 immobilized pH gradients. The genome of the human gut pathogen, Helicobacter pylori, is dominated by genes encoding basic proteins, and is therefore a useful model for examining methodology suitable for separating such proteins. H. pylori proteins were separated on pH 6-11 and novel pH 9-12 immobilized pH gradients and 65 protein spots were subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry, leading to the identification of 49 unique proteins. No proteins were characterized with a theoretical pI of greater than 10.23. A second approach to examine extremely alkaline proteins (pI > 9.0) utilized a prefractionation isoelectric focusing. Proteins were separated into two fractions using Gradiflow technology, and the extremely basic fraction subjected to both sodium dodecyl sulphate-polyacrylamide gel electrophoresis and liquid chromatography (LC) - tandem mass spectrometry post-tryptic digest, allowing the identification of 17 and 13 proteins, respectively. Gradiflow separations were highly specific for proteins with pI > 9.0, however, a single LC separation only allowed the identification of peptides from highly abundant proteins. These methods and those encompassing multiple LC 'dimensions' may be a useful complement to 2-DE for 'near-to-total' proteome coverage in the alkaline pH range.     

Prefractionation of proteome by liquid isoelectric focusing prior to two-dimensional liquid chromatography mass spectrometric identification

Due to the complexity of proteomes, developing methods of sample fractionation, separation, concentration, and detection have become urgent to the identification of large numbers of proteins, as well as the acquisition of those proteins in low abundance. In this work, liquid isoelectric focusing (LIEF) combined with 2D-LC-MS/MS was applied to the proteome of Saccharomyces cerevisiae. This yielded a total of 1795 proteins that were detected and identified by 30 fractions of protein prefractionation. Categorization of these hits demonstrated the ability of this technology to detect and identify proteins rarely seen in proteome analysis without protein fractionation. LIEF-2D-LC-MS/MS also produced improved resolution of low-abundance proteins. Furthermore, we analyzed the characteristics of proteins obtained by LIEF-2D-LC-MS/MS. 1103 proteins with CAI under 0.2 were identified, allowing us to specifically obtain detailed biochemical information on these kind proteins. It was observed that LIEF-2D-LC-MS/MS is useful for large-scale proteome analysis and may be specifically applied to systems with wide dynamic ranges.     

The Characterization of Tubulin in CNS Membrane Fractions

Abstract— Rough endoplasmic reticulum (RER), smooth endoplasmic reticulum (SER), and a plasma membrane (PM) fraction enriched in synaptic membranes were isolated from rat forebrain. The proteins in these membrane fractions were analyzed by two-dimensional gel electrophoresis (2DGE) in the isoelectric range of 5.1 to 6.0 by a modification of the O'Farrell procedure. Proteins were detected by Coomassie Brilliant Blue staining of the electrophoretograms. The results of these analyses were compared with 2DGE analysis of cytosol proteins, with particular attention given to tubulin subunits and actin. The RER contained one major protein (53K 5.4) in the β-tubulin region with a molecular weight of 53,000 and an isoelectric point of 5.4. The SER contained at least two major proteins in the β-tubulin region; one with a migration identical to 53K 5.4 and other proteins with slightly higher apparent molecular weights and more acidic isoelectric points (54K, 5.4 to 5.3), identical to cytoplasmic β-tubulin. The PM fraction also contained multiple overlapping proteins (54K, 5.4 to 5.3) in the β-tubulin area and a trace amount of the 53K 5.4 protein. The proteins in the β-tubulin region were removed from the 2DGE electrophoretogram and digested by Staphylococcus aureus V8 protease, and the peptides separated on one-dimensional polyacrylamide gels. The peptide patterns of 53K 5.4 protein from RER and SER were almost identical and differed significantly from the cytoplasmic β-tubulin pattern; however, the peptide maps of the PM and SER β-tubulin region were identical to the cytoplasmic β-tubulin. The 2DGE analysis of RER did not contain proteins in the region of cytoplasmic α-tubulin. SER and PM contained proteins in the α-tubulin region with a similar, but not identical, peptide analysis to cytoplasmic α-tubulin. Significant amounts of actin were detected in 2DGE analysis of SER and PM, and the peptide analysis of the actin was identical to the cytoplasmic actin analysis. The RER fraction contained only trace amounts of actin. The cytosol and all membrane fractions contained a protein (68K 5.6) found among microtubule-associated proteins, as judged by molecular weight and isoelectric point. Several proteins present in all membrane fractions (61K 5.1 and 58K 5.1) bound to concanavalin A agarose.     

Sequence information and preparation of resolved by; two-dimensional gel electrophoresis: no longer just spots on gels

In 1975 O'Farrell described, in detail, a procedure to separate proteins by polyacrylamide gel electrophoresis in two dimensions. This powerful new technique relied on two characteristics of proteins: charge and molecular mass. In the first dimension, proteins were separated on the basis of net charge in a pH gradient by isoelectric focusing, and in the second dimension the proteins were further fractionated exclusively on the basis of their molecular mass by SDS gel electrophoresis. Since two-dimensional gel electrophoresis (2DGE) has a resolving power of at least 20 fold greater than one-dimensional electrophoresis, it has found wide spread application in modern biological research. However, beyond the detection of a given protein, 2DGE provides little additional information about a specific protein other than molecular mass, isoelectric point, and approximate relative abundance. In recent years, the development of new technologies have made it possible to directly obtain sequence information, and produce specific antisera for proteins resolved by 2DGE. These new technological developments serve to further increase the power and utility of 2DGE in the analysis of proteins of importance to plant physiology.     

Depletion of hemoglobin and carbonic anhydrase from erythrocyte cytosolic samples by preparative clear native electrophoresis

Proteomic analysis of red cells is compromised by the presence of high-abundance proteins (hemoglobin and carbonic anhydrase-1), which completely obscure low-abundance species. The depletion method presented here involves performing native gel electrophoresis in a polyacrylamide gel tube using a modified electroelution cell. The electrophoretic run is interrupted intermittently to allow the recovery of at least three different liquid fractions, which can be analyzed by both native PAGE and 2D isoelectric focusing SDS-PAGE, or by shotgun mass spectrometry analysis after trypsin in-solution protein digestion. This low-cost, reproducible technique can be used to process large amounts of sample, and it increases the likelihood of detecting low-abundance proteins, thereby resulting in greater proteome coverage. The separation procedure takes approximately 6-7 h.     

pI-based fractionation of serum proteomes versus anion exchange after enhancement of low-abundance proteins by means of peptide libraries

The pre-treatment of biological extracts with the aim of detecting very low-abundance proteins generates complexity requiring a proper fractionation. Therefore the success of identifying all newly detectable species depends on the selected fractionation methods.In this context and starting from a human serum, where the dynamic concentration range was reduced by means of a preliminary treatment with a combinatorial hexapeptide ligand library, we fractionated the sample using a novel method based on the differences in isoelectric points of proteins by means of Solid-State Buffers (SSB) associated with cation exchangers. The number of fractions was limited to four and was compared to a classical anion exchange method generating the same number of fractions.What was observed is that when using SSB technology the protein redundancy between fractions was significantly reduced compared to ion exchange fractionation allowing thus a better detection of novel species. The analysis of trypsinized protein fractions by nanoLC-MS/MS confirmed that the SSB technology used is more discriminant than anion exchange chromatography fractionation.A sample fractionation by SSB after the reduction of dynamic concentration range can be accomplished without either adjustment of pH and ionic strength or protein concentration and cleanup. Both advantages over either classical chromatography or isoelectric fractionations allow approaching the discovery of markers of interest under easier conditions applicable in a variety of fields of investigation.     

Parallel isoelectric focusing chip

Fast isoelectric focusing (IEF) is becoming a key method in modern protein analysis. We report here the theory and experimental results of new parallel isoelectric devices (PID) for fast IEF. The main separation tool of any PID is a dielectric membrane with conducting channels filled by immobiline gels of varying pH. The pH value of the surrounding aqueous solution is not equal to the pH of any of the channels. The membrane is held perpendicular to the applied electric field. Proteins are collected (trapped) in the channels whose pH values are equal to the pI of the proteins. The fast particle transport between different channels takes place due to convection in the aqueous solution. We developed a mathematical model for PID. Experiment duration is shown to be proportional to the number of different bands N (the peak capacity in standard IEF) in contrast with N(2) for usual IEF devices. This model was validated with experimental results. Parallel IEF accelerates the fractionation of proteins by their pI values (down to several minutes) allowing a more desirable collection efficiency to be achieved. The main theoretical limitation of PID resolution is the sensitivity of proteins to pH change due to the Coulomb blockade effect. The existence of a minimal pH change deltapH(min) for each type of protein is shown: deltapH(min) approximately r(-1) for globular molecules with radius r.     

Biosynthesis of the common precursor to vasopressin and neurophysin in vitro in transplantable human oat cell carcinoma of the lung with ectopic vasopressin production

Transplantable human oat cell carcinoma cells of the lung with ectopic vasopressin production were incubated with labeled amino acids and immunoreactive neurophysins in cell extracts were analyzed by isoelectric focusing. When the cells were incubated with L-(35S)-cysteine for 20 h, one major peak (isoelectric point; pI=5.3) and several minor peaks (pI=6.1, 5.7, 5.1, 4.9 and 4.7) of labeled proteins were observed. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the relative molecular mass (Mr) of the pI 5.7 protein was estimated to be 20,000 and that of the pI 6.1 species to be 19,000, while the remainder had a Mr of approximately 10,000. The result of the pulse-labeling experiment has clearly shown that the pI 5.7 and 6.1 proteins, which have affinity for concanavalin A, are biosynthetic precursors for the smaller form of neurophysin with a pI 5.3. When subjected to limited proteolysis with trypsin, the pI 5.7 protein generated a Mr 10,000 protein and a smaller peptide. The Mr 10,000 protein thus produced was identified as neurophysin on the basis of its pH-dependent affinity for vasopressin and the migration pattern on isoelectric focusing. The smaller peptide coeluted with synthetic arginine vasopressin and bound to neurophysin suggesting that it possesses a cysteine-tyrosyl sequence at its N-terminus. Similarly, the pI 6.1 protein liberated neurophysin and vasopressin-like peptide after incubation with trypsin. These results suggests that the glycosylated protein with a pI of 5.7 and a Mr of 20,000 is the common precursor to vasopressin and neurophysin in human oat cell carcinoma of the lung with ectopic vasopressin production. The pI 6.1 protein may be an intermediate in the conversion of the precursor to vasopressin and neurophysin.     

Analysis of rat serum apolipoproteins by isoelectric focusing. II. Studies on the low molecular weight subunits.

The low molecular weight proteins of rat apo HDL and apo VLDL have been isolated and analyzed by the technique of isoelectric focusing. Sephadex fractions from apo HDL (HS-3) and apo VLDL (VS-3) that contain these proteins reveal three major bands with apparent isoelectric points of pH 4.50, 4.67, and 4.74, as well as three minor bands at pH 4.43, 4.57, and 4.61. In addition, apo HDL has a major band at pI of 4.83. DEAE-Cellulose chromatography was used to prepare purified fractions of these components that were characterized by N-terminal analyses and molecular weight determinantions by SDS gel electrophoresis. The major low molecular weight components of apo HDL were focused on a slab gel and the bands were identified as A-II (pI 4.83), C-II (pI 4.74), C-III-0 (pI 4.67), and C-III-3 (pI 4.50). Neuraminidase treatment of apo HDL, followed by isoelectric focusing, suggested that the other bands, which have not previously been reported, may be additional forms of the C-III protein, differing only in their content of sialic acid.     

Advances in the Purification of the Mitochondrial Ca2+ Uniporter Using the Labeled Inhibitor 103Ru360

For many years the calcium uniporter has eluded attempts of purification, partly because of the difficulties inherent in the purification of low-abundance hydrophobic proteins (Reed and Bygrave, 1974). Liquid-phase preparative isoelectric focusing improved the fractionation of mitochondrial membrane proteins. A single 6-h run resulted in a 90-fold increase in specific activity of pooled active fractions over a semipurified fraction, allowing for enrichment of the calcium transport function in cytochrome oxidase vesicles. An additional powerful tool in the isolation of the uniporter was the use of the labeled inhibitor ¹⁰³Ru₃₆₀ as an affinity ligand; by following this procedure a protein of 18 kDa was purified in nondenatured, but rather inactive, form. The labeled protein corresponds to the protein that showed Ca²⁺ transport activity.     

The vitamin K-dependent bone protein is accumulated within cultured osteosarcoma cells in the presence of the vitamin K antagonist warfarin

Osteosarcoma cells grown in normal culture medium secrete bone gamma-carboxyglutamic acid protein (BGP, osteocalcin) which is identical with BGP purified from the bone matrix. Two tests indicate that the secreted medium protein contains the full complement of three gamma-carboxyglutamate residues present on BGP purified from the bone matrix. First, the secreted protein from ROS 17/2 and bone matrix BGP have identical isoelectric points (pI = 4.0). Second, they have identical hydroxyapatite binding behavior. If warfarin is added to the culture medium, the secreted protein has a higher isoelectric point (pI = 4.6) and a lower affinity for hydroxyapatite characteristic of thermally decarboxylated or non-gamma-carboxylated BGP. The observed shift in isoelectric point of secreted BGP after warfarin treatment from pH 4.0 to 4.6 is also reflected in the presence of pI = 4.1 and pI = 4.6 species intracellularly. These isoelectric species correspond to fully carboxylated BGP and noncarboxylated BGP, which are in the process of secretion. Addition of 10 micrograms/ml of warfarin causes a specific 47% reduction in secretion rate of BGP, while at the same time, the intracellular BGP concentration increases 3-fold. These phenomena appear related to the interruption by warfarin of the normal sequence of processing of precursor BGP proteins, as a new, immunoreactive species with a higher isoelectric pH not present in control cells appears to be responsible for the increased intracellular antigen within warfarin-treated cells. Our results show that vitamin K-dependent processing is important for normal secretion of BGP from the cell.     

Isoelectric protein purification in segmented immobilized pH gradients. Effect of salts on rate of contaminants' removal

A method is described for keeping a constant salt background during protein purification in a segmented immobilized pH gradient. It is based on an external hydraulic flow replenishing the salt loss due to combined electric and diffusional mass transport (similar to the concept of Ribes' steady-state rheoelectrolysis). Such a minimum of ionic strength might be needed for proteins which tend to precipitate and aggregate at or in vicinity of the isoelectric point. However, it is found that any salt level in the sample feed (already at 1 mM concentration) deteriorates transport of non-isoelectric proteins, because of the much larger current fraction carried by the ions themselves as opposed to proteins. In addition, high salt levels in the sample reservoir might form cathodic and anodic ion boundaries, alkaline and acidic, respectively, which might hamper protein migration and even induce denaturation. Thus, when high salt backgrounds are needed in the sample feed, external pH control should be exerted, e.g. with a pH-stat. Three parameters influence protein transport in the segmented IPG chamber: (a) cross-sectional area of the Immobiline membranes; (b) delta pI between the isoelectric protein and the contaminants and (c) salt molarity in the sample reservoir. The first 2 show a positive, the last a negative correlation.     

De novo synthesis of peroxidase in cotton ovule culture medium

The biosynthesis of cotton ( Gossypium hirsutum L. 'Stoneville 208') peroxidase (EC 1.11.1.7) has been investigated in an organ culture system, since this enzyme may play a role in cell wall biogenesis or host defense mechanisms. Electrophoretic analysis of proteins from cotton ovule cultures indicated relatively few proteins being released into the surrounding medium. De novo synthesis of released peroxidase and other medium proteins was determined by in vivo labeling of ovule cultures with [³⁵S]-methionine. Analysis of labeled culture medium by denatured gel electrophoresis followed by fluorography showed incorporation of isotope into 2 major proteins with molecular weights of 30 kD and 56 kD, as well as a limited number of minor proteins. Similar analysis of native isoelectric focusing gels coupled with autoradiography demonstrated [³⁵S]-methionine incorporation into 2 major proteins with pI values of 4.3 and 5.0. The pI 5.0 protein was shown to have a molecular weight of 30 kD. The pI 4.3 protein had a molecular weight of 56 kD and was shown to be peroxidase by activity staining. Minor radiolabeled proteins were observed in the cationic region of the isoelectric focusing gels.     

Studies on the Biosynthesis of Intermediate Filament Proteins in the Rat CNS

Abstract: The biosynthesis of brain intermediate filament proteins [neurofilament proteins and glial fibrillary acidic protein (GFA)] was studied with cell-free systems containing either rat spinal cord polysomes (free polysomes or rough microsomes) and rabbit reticulocyte factors or wheat germ homogenate containing spinal cord messenger RNA. The products of translation were isoated by immunoaffinity chromatography and then analyzed by two-dimensional gel electrophoresis (2DGE) followed by fluorography. The free polysome population was found to synthesize two neurofilament proteins (MW 145K, p15.4, and MW 70K, pl 5.3) and three isomers of GFA (α, β, and γ) that differ in isoelectric point. Wheat germ homogenate containing messenger RNA extracted from free cord polysomes synthesized two proteins that comigrated with neurofilament protein standards at 145K 5.4 and 70K 5.3; these proteins were partially purified by neurofilament affinity chromatography. The wheat germ system also synthesized the α, β, and γ isomers of GFA as characterized by immunoaffinity chromatographic purification and comigration with standards in 2DGE analysis. Our data are consistent with the conclusion that synthesis of neurofilament proteins requires multiple messenger RNAs. Also, synthesis of intermediate filament proteins occurs in the free polysome population; detectable amounts of these proteins were not synthcsized by the rough microsomes.     

Susceptibility of muscle soluble proteins to degradation by mast cell chymase

We investigated the in vitro susceptibility of muscle soluble proteins to the major alkaline proteinase (chymase) from skeletal muscle tissue, an enzyme originating from intramuscular mast cells, but also present in certain muscle fibers. Cytoplasmic proteins from rat skeletal muscle tissue were fractionated into four groups according to their different isoelectric points: fraction A (pI 9.5-7.0), B (pI 7.0-5.6), C (pI 5.5-4.5) and D (pI 5.3-3.5). Chromatography of these fractions on octyl-Sepharose CL-4B revealed the presence of a higher percentage of hydrophobic proteins in fraction C and D as compared to fraction A and B. In vitro degradation of these protein fractions by chymase, isolated from rat skeletal muscle tissue, was monitored (a) by measuring the ability of these proteins to bind Coomassie G-250, and (b) by analyzing the digestion mixture in isoelectric focusing gels. Both methods revealed fraction B proteins to be degraded very rapidly. While there was also a significant breakdown of fraction A proteins, fraction C and D proteins were degraded only very slowly, if at all. These differences in degradability are not due to the presence of a proteinase inhibitor in fraction C and D. The results suggest that mast cell chymase preferentially degrades those groups of muscle soluble proteins, the constituents of which have neutral to basic isoelectric points and a relatively low surface hydrophobicity.     

Protein purification by Off-Gel electrophoresis

A novel free-flow protein purification technique based on isoelectric electrophoresis is presented, where the proteins are purified in solution without the need of carrier ampholytes. The gist of the method is to flow protein solutions under an immobilised pH gradient gel (IPG) through which an electric field is applied perpendicular to the direction of the flow. Due to the buffering capacity of the IPG gel, proteins with an isoelectric point (pI) close to pH of the gel in contact with the flow chamber stay in solution because they are neutral and therefore not extracted by the electric field. Other proteins will be charged when approaching the IPG gel and are extracted into the gel by the electric field. Both a demonstration experiment with pI markers and a simulation of the electric field distribution are presented to highlight the principle of the system. In addition, an isoelectric fractionation of an Escherichia coli extract is shown to illustrate the possible applications.     

Isoelectric point-based prefractionation of proteins from crude biological samples prior to two-dimensional gel electrophoresis

Two-dimensional gel electrophoresis (2-DE) is used to compare the protein profiles of different crude biological samples. Narrow pH range Immobilized pH Gradient (IPG) strips were designed to increase the resolution of these separations. To take full advantage of IPG strips, the ideal sample should be composed primarily of proteins that have isoelectric point (pI) values within the pH range of the IPG strip. Prefractionation of cell lysates from a human prostate cancer cell line cultured in the presence or absence of epigallocatechin-3-gallate was achieved in fewer than 30 min using an anion-exchange resin and two expressly designed buffers. The procedure was carried out in a centrifuge tube and standard instrumentation was used. The cell lysates were prefractionated into two fractions: proteins with pI values above 7 and between 4 and 7, respectively. The fractions were then analyzed by 2-DE, selecting appropriate pH ranges for the IPG strips, and the gels were compared with those of unprefractionated cell lysates. Protein loading capacity was optimized and resolution and visualization of the less abundant and differentially expressed proteins were greatly improved.     

Two-dimensional fluorescence difference gel electrophoresis for comparative proteomics profiling

Quantitative proteomics is the workhorse of the modern proteomics initiative. The gel-based and MuDPIT approaches have facilitated vital advances in the measurement of protein expression alterations in normal and disease phenotypic states. The methodological advance in two-dimensional gel electrophoresis (2DGE) has been the multiplexing fluorescent two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). 2D-DIGE is based on direct labeling of lysine groups on proteins with cyanine CyDye DIGE Fluor minimal dyes before isoelectric focusing, enabling the labeling of 2-3 samples with different dyes and electrophoresis of all the samples on the same 2D gel. This capability minimizes spot pattern variability and the number of gels in an experiment while providing simple, accurate and reproducible spot matching. This protocol can be completed in 3-5 weeks depending on the sample size of the experiment and the level of expertise of the investigator.